The present invention relates to the crystalline structure of isoleucyl-tRNA synthetase and the cognate tRNAile and to methods of producing such crystals. The invention also relates to the atomic coordinates of isoleucyl-tRNA synthetase and the cognate tRNAile, obtained by x-ray diffraction at high resolution. The present invention also relates to methods for identifying and designing new classes of ligands which target the isoleucyl-tRNA synthetases of specific organisms. The methods and compositions of the present invention find wide applicability in the design and production of antibiotics, insecticides, miticides and herbicides.
Mupirocin
The most important invention in medicine in this century is perhaps the discovery of penicillin by Alexander Fleming in 1928, a naturally occurring antibiotic that inhibits cell-wall synthesis in many pathogenic bacteria. In 1940, E. B. Chain and H. W. Florey were able to produce stable commercial formulations of this antibiotic. For this invention, Fleming, Chain, and Florey shared the Nobel Prize in medicine or physiology in 1945.
In the past half century, from penicillin to methicilin to vancomycin, over 130 related antibiotics have been discovered that inhibit cell-wall synthesis (Neu, 1991). The art of the discovery is relatively simple; it requires simply a combination of microbiology and organic chemistry. Any organic chemical that inhibits bacterial cell growth by acting on cell-wall synthesis are good antibiotics, since only bacteria, not human cells, have cell wall. In comparison, the same approach that has worked for the discovery of antibiotics that inhibit cell-wall synthesis has not worked well for the discovery of antibiotics that inhibit protein synthesis.
The antibiotic for inhibition of protein synthesis, pseudomonic acid, remains in its original form since it was first discovered about three decades ago by E. B. Chain and his colleagues (Fuller et al., 1971). However, it has been since renamed as mupirocin. Mupirocin is the active ingredient of Bactroban(trademark), a trademark of SmithKline Beecham. All attempts so far have failed to modify this antibiotic with either improved stability against unknown human hydrolase(s) for in vivo use or improved selectivity for its pathogenic target enzyme over human enzyme, simply because no organic chemists know how to modify the antibiotic to achieve the above goals.
Staphylococcus aureus (SA), present in about two-thirds of healthy individuals in the entire population, has a long association with nosocomial infection and is a virulent pathogen that is currently the most common cause of infections in hospitalized patients (Archer, 1998, Gould and Chamberlaine, 1995). In 1941, virtually all strains of S. aureus worldwide were susceptible to penicillin G, the first antibiotic used in clinics, but by 1944, S. aureus began to become resistant to the antibiotic, and by late 1980s, more than 95% of S. aureus worldwide were resistant to penicillin, amplicillin, and the antipseudomonas penicillins (Lyon and Skurray, 1987). In response, the pharmaceutical industry produced a second generation antibiotic, methicillin, a semisynthetic penicillin. However, methicillin-resistant S. aureus (MRSA) became a severe problem in the 1980s (Vandenbrouche-Grauls, 1994, Mulligan et al., 1993), and is resistant to all xcex2-lactams because it produces a new penicillin binding protein to remove all related antibiotic, pencillins, cephalosporins, carbapenems, and penems (Lyon and Skurray, 1987; Ubukata et al., 1985; Murakami and Tomasz, 1989; Tesch et al., 1988; Chambers and Sachdeva, 1990). The emergence of MRSA as a major problem worldwide has resulted in an increased use of vanomycin, the only effective antibiotic and often reserved for use in patients who are gravely ill. Its increased use has created vancomycin-resistant pathogens including S. aureus (Flores and Gordon, 1997, Perl, 1999, Paterson, 1999, Neu, 1992).
Mupirocin, a derivative of pseudomonic acid from Pseudomona fluorescens (Fuller et al., 1971), is highly effective against MRSA (Bertino, 1997, Dacre et al., 1986). Differing from cell wall-inhibiting antibiotic, it binds isoleucyl-tRNA synthetase (IRS) as a competitive inhibitor for isoleucine and inhibits protein biosynthesis (Hughes and Mellows, 1978ab; Hughes and Mellows, 1980; Yanagisawa et al., 1994; Pope et al., 1998ab). Topical use of mupirocin has very successfully eradicated the nasal carriage of MRSA (Harbarth et al., 1999; Redhead et al., 1991; Casewell and Hill, 1989; Caderna et al., 1990). This is extremely important because the anterior opening to the nasal cavities (i.e., the naris or nares), are the major site where MRSA and susceptible staphylococci persist. Topical use also eradicated MRSA in skin and virginal infections after the failure of intervenous vancomycin therapy (Denning and Haiduven-Griffiths, 1988, Cool-Foley et al., 1991). Despite these success, a person could still die in a hospital in any major city with a resistant bacterial infection. Although mupirocin resistant S. aureus (MURSA) is rare, it exists (Anthony et al., 1999; Schmitz et al., 1998; Gilbart et al., 1993; Farmer et al., 1992; Capobianco et al., 1989; Eltringham, 1997; Woodford et al., 1998).
Mupirocin is not very effective against bacteremia caused by MRSA because of its short half-life metabolic conversion in vivo from pseudomonic acid to inactive monic acid, which is rapidly cleared in the urine (Mellows, 1989). The pharmaceutical industry has been unsuccessful in slowing or halting the enzymatic hydrolysis by modifying the structure and function of the C1-C3 fragment, although the modified antibiotic retains good in vitro activity (Rogers 1980, Rogers and Coulton, 1882, Banks et al., 1989). This fragment has also been replaced by an unsaturated 5-membered heterocycle ring, but it must retain low-energy unoccupied molecular orbital for its inhibitory activity (Brown et al., 1997).
Selectivity of Isoleucyl-tRNA Synthetase
The high fidelity of genetic information transfer in translation is essential for the survival of organisms. Translation accuracy depends on the ability of amino acid tRNA synthethases to discriminate among tRNAs and among amino acids in amino acylation. Discrimination of L-isoleucine over L-valine by isoleucyl-tRNA synthetase is one of most difficult recognitions to achieve, because L-isoleucine and L-valine differ by only one methylene group in their aliphatic side chains. Additionally, this enzyme is the target of mupirocin, the only effective antibiotic that inhibits protein synthesis. This enzyme has therefore been extensively studied in over a half century, leading to the present invention (Silvian et al., 1999).
Isoleucyl-tRNA synthetase (IRS) selectively adds isoleucine to isoleucyl-tRNA, while rejecting all other amino acids and all other noncognate tRNAs. This enzymatic selectivity of isoleucine over valine is over 3000-fold (Loftfield, 1963; Loftfiled and Vanderjagt, 1972). If IRS were an inorganic catalyst, a free energy difference of one single methylene group between the two amino acids would provide only about 5-fold difference in selectivity (Pauling, 1958) based on an adsorption theory, which has successfully explained catalytic mechanisms for nearly all inorganic catalysts. According to the theory, inorganic catalysts (such as transition metal ions) accelerate rates of chemical reactions by increasing the collision frequency through adsorbing two reactants on catalysts"" surface. The rate of enhancement is a function of adsorption of free energy, and the selectivity of given reactions is a function of free energy differences in the adsorption. A large discrepancy in selectivity of the synthetase led Baldwin and Berg (1966) to discover the hydrolytic activity of the enzyme and led Dingwall and Fersht (1979a,b) to propose a xe2x80x9cdouble-sievexe2x80x9d hypothesis. This hypothesis predicted that there are two distinct enzymatically active sites, one hydrolytic and one synthetic. Amino acids that are larger than isoleucine are rejected by steric exclusion in the first sieve in the synthetic active site. Amino acids that are smaller than isoleucine fit into the second sieve in the hydrolytic active site and are rejected by hydrolysis.
Hydrolysis by IRS includes two substrates, both of which are the IRS synthetic products. One is an incorrectly acylated valine-tRNAile, also known as a post-transfer product, and the other, the dominant substrate for hydrolysis, is an activated noncognate valine-adenylate, also known as a pre-transfer product (Fersht, 1977). IRS is a model system for mechanistic studies of editing tRNA synthetases (Freist, 1989). Nureki et al. recently (1998) determined the IRS crystal structure from T. thermophilus (Tth) and showed that indeed there are two active sites, located in two distinct domains, separated by over 34 xc3x85, providing direct evidence for the double-sieve hypothesis.
There is a parallelism between IRS and DNA polymerases. Komberg and his colleagues (Setlow et al., 1972, Brutlag and Komberg, 1972) discovered the hydrolytic activity in DNA polymerases, where an adsorption theory on the basis of base-pairing, which could only provide about 35-50 fold selectivity (Johnson, 1993), failed to explain the observed selectivity. The crystal structure of the Klenow fragment of E. coli DNA polymerase I (Ollis et al., 1985) showed that the exonuclease (hydrolytic) and the polymerase (synthetic) do not share a single active site. They are located in two distinct domains, separated by over 30 xc3x85, a surprising result that was not anticipated by biochemical data at that time (Huberman and Kornberg, 1970). Further, the crystal structure showed that the two activities are independent of each other and can be physically separated (Freemont et al., 1986).
Crystals of IRS
Nureki et al. (1998) discloses the crystal structure of T. thermophilus IRS complexed to L-isoleucine or L-valine. The crystal structure has a resolution of 2.4 xc3x85 and is obtained by analysis of X-ray crystallographic diffraction data of the crystal. The crystal of Nureki et al. (1998) show that the first step in substrate selection is on the aminoacylation domain containing.the Rossmann fold, whereas the second step, the editing step, exists on a globular xcex2-barrel domain that protrudes from the amino acylation domain. The structures of the isoleucyl-tRNA synthetases from S. aureus and T. thermophilus are homologous, and the active sites of these synthetases are also structurally similar. However, the crystal structure of Nureki et al. (1998) does not include structural information for mupirocin or tRNAile and how mupirocin, other protein synthesis inhibitors, or tRNAile interacts with T. thermophilus IRS.
The present invention discloses the crystal structure of S. aureus IRS complexed to muciprocin and tRNAile. The present invention shows how IRS interacts with an inhibitor of protein synthesis, in the presence of tRNAile.
The present invention provides methods of preparing crystals of a complex comprising isoleucyl-tRNA synthetase (IRS) complexed with mupirocin, and tRNAile which includes mixing IRS, mupirocin, and tRNAile with a well solution to form a mixture; streak-seeding drops of the mixture; vapor equilibrating the seeded drops in a closed container against the well solution to obtain a crystal of the complex and to produce an equilibrated crystal drop solution; replacing the equilibrated crystal drop solution with a cryoprotectant; and flash-freezing the crystal. More particularly the present inventions provides such methods wherein the well solution comprises about 12% PEG 6K, about 0.3 M KCl, about 100 mM Na Cacodylate pH 6.3, about 100 mM MgSO4, about 2 mM ZnCl2 and about 0.1% xcex2-octyl glutopyranoside. The present invention also provides such methods wherein the seeded drops are equilibrated by hanging drop method. The present invention further provides such methods wherein the cryoprotectant comprises about 20% PEG 6K, about 0.3 M KCl, about 100 mM Na Cacodylate pH 6.3, about 100 mM MgSO4, about 2 mM ZnCl2 about 0.1% xcex2-octyl glutopyranoside, and about 15% ethylene glycol. The present invention also provides such methods wherein the crystal is flash-frozen in liquid propane.
The present invention also provides crystals of IRS, mupirocin and tRNAile. More particularly, the present invention provides such crystals wherein the crystals effectively diffract X-rays for determination of atomic coordinates of the complex to a resolution of about 2.2 xc3x85. Even more particularly, the present invention provides such crystals wherein the crystals have two unit cell sizes, wherein the first unit cell comprises of the dimensions a=71 xc3x85, b=100 xc3x85 and c=186 xc3x85 and wherein the second unit cell has the dimensions a=71 xc3x85, b=100 xc3x85 and c=180 xc3x85. In addition, the present invention provides such crystals wherein the crystals belong to the space group P2l2l2l.
The present invention provides crystals which have an atomic structure characterized by the coordinates deposited at the Protein Data bank with accession number PDB ID: 1FFY.
In particular, the present invention provides the above-listed crystals which are obtained from Staphylococcus aureus. 
The present invention also provides methods for identifying agents (ligands) that interact with IRS and tRNAile, wherein such methods include obtaining a crystal of a complex comprising IRS, tRNAile and mupirocin; obtaining the atomic coordinates of the crystal; and using the atomic coordinates and one or more molecular modeling techniques to identify an agent that interacts with IRS and tRNAile.
The present invention further provides methods of identifying agents (ligands) that interact with IRS wherein such methods include obtaining a crystal of a complex comprising IRS, tRNAile and mupirocin; obtaining the atomic coordinates of the crystal; and using the atomic coordinates and one or more molecular modeling techniques to identify an agent that interacts with IRS.
More particularly, the present invention provides such methods of identifying agents (ligands) wherein the one or more molecular modeling techniques include graphic molecular modeling and computational chemistry.
The present invention further provides such methods of identifying agents (ligands) and then contacting the agents with IRS and detecting the amount and degree of binding of the agents to IRS. The methods of the present invention can be used to identify agents that bind to enzymes from the same or different species as the species from which the enzyme was obtained to produce the crystal.
The present invention further provides such methods of identifying agents which include altering the identified agents and contacting the altered agents with IRS and determining the binding of the altered agents to IRS.
The present invention also provides altered agents produced by such methods wherein the altered agents bind differently to IRS than do the agents from which the altered agents were derived. The present invention further provides such altered agents wherein the altered agents are therapeutic agents. More particularly, the present invention provides such altered agents wherein the altered agents have desirable pharmaceutical properties. The invention further provides compositions which include such altered agents combined with pharmaceutically-acceptable carriers.
More particularly, the present invention provides altered agents which act as inhibitors of IRS. The agents of the present invention can be specifically designed to kill or act as inhibitors of target organisms while not killing non-target organisms or inhibiting non-target organisms less at the same concentration of the altered agents.
The present invention also provides methods of identifying inhibitors of protein synthesis which include obtaining crystals of a complex comprising IRS, tRNAile and mupirocin; obtaining the atomic coordinates of the crystals; using the atomic coordinates and molecular modeling techniques to identify agents that interact with IRS; assaying the inhibitory properties of the agents by administering them to cells, cell extracts or purified IRS; and detecting protein synthesis, wherein a decrease in protein synthesis indicates that the agents are inhibitors of protein synthesis. The present invention also provides such methods wherein assaying the inhibitory properties of the agents includes detecting protein synthesis and wherein a decrease in protein synthesis indicates that the agents are inhibitors of protein synthesis. The present invention also provides such methods wherein assaying the inhibitory properties of the agents includes determining an inhibition constant for inhibiting isoleucyl-tRNA synthesis reaction by the agents.
The present invention also provides methods of identifying inhibitors of protein synthesis which include obtaining crystals of a complex comprising IRS, tRNAile and mupirocin; obtaining the atomic coordinates of the crystals; using the atomic coordinates and molecular modeling techniques to identify agents that interact with IRS; and assaying the inhibitory properties of the agents by administering them to cells, a cell extracts or purified IRS to determine whether they are inhibitors of protein synthesis. The present invention also provides such methods wherein assaying the inhibitory properties of the agents includes determining whether the agents inhibit isoleucyl-tRNA synthesis. More particularly, the present invention provides such methods wherein whether the agents inhibit isoleucyl-tRNA synthesis are determined by measuring the generation of pyrophosphate or the formation of isoleucyl-tRNAile.